The present invention relates generally to a method of amplifying closed circular nucleic acid probes and, more particularly, to a method of amplifying closed circular nucleic acid probes by rolling circle amplification. The method of the present invention is useful in a range of applications involving the detection of nucleic acid sequences such as, but not limited to, the identification of genetic disorders, genetic variants or the presence of microbiological or viral agents.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
A variety of nucleic acid amplification technologies exist for the diagnosis of infectious and genetic diseases. Since its invention over a decade ago, the polymerase chain reaction (PCR) (1) has become the method of choice in research and DNA-based diagnostics. This can be attributed to its speed, simplicity and sensitivity. PCR does, however, require temperature cycling, which therefore necessitates the use of expensive thermal cycling equipment. Other amplification techniques, which also require temperature cycling, include the ligase chain reaction (LCR) (2) and the transcription-based amplification system (TAS) (3).
Various other amplification techniques exist which do not require extensive thermal cycling and are essentially isothermal systems. Several of these are transcription-medited or require RNA as an integral component of the reaction therefore necessitating that the amplification environment is kept free from ribonuclease contamination. These methods include the Qxcex2 replicase system (4), self-sustained sequence replication (3SR) (5) and nucleic acid sequence-based amplification (NASBA) (6).
Presently, there appear to exist at least two isothermal techniques for the amplification of nucleic acid sequences which essentially do not require RNA intermediates. Strand displacement amplification (SDA) (7) is an isothermal technique which relies on the ability of a restriction enzyme to nick a hemiphosphorothioated recognition site and the ability of a polymerase to initiate replication at a nick and displace the downstream strand. The other isothermal technique which can be used to amplify a nucleic acid sequence is rolling circle amplification (RCA).
Various forms of the rolling circle amplification technique have previously been described (8,9). In essence the technique relies on amplification from a circular DNA probe. The circular probe, commonly referred to as a xe2x80x9cpadlock probexe2x80x9d, is designed such that it has regions at both its 5xe2x80x2 and 3xe2x80x2 ends which are complementary to the target sequence of interest and are separated by a region of nucleotide of non-target derived origin. Upon hybridisation, the 5xe2x80x2 and 3xe2x80x2 ends of the probe are brought into ciose proximity to one another. If the two probe regions are adjacent to one another the 5xe2x80x2 and 3xe2x80x2 ends can be joined to produce a circular probe, In some instances, however, the probe regions are separated from one another by a small stretch of nucleotides. This region must be filled to achieve the generation of a circular probe. In this regard, a variety of techniques can be utilised including the use of spacer oligonucleotides or by using a DNA polymerase (or a reverse transcripts in the case of an RNA target) in combination with deoxynucleotide triphosphate molecules to fill the gap prior to ligation.
A significant problem associated with the rolling circle amplification technique is the occurrence of background amplification. Prior to the advent of the present invention this background amplification was dismissed as primer-induced deletion fragment repeats encompassing a full unit repeat minus the intervening region between 5xe2x80x2 ends of the two primers (8). Background amplification represents both a significant problem and a limitation for rolling circle amplification reactions which utilise 2 primers. It is also a major source of false positive results. In fact, the magnitude of the problem presented by the occurrence of this background amplification bas been such that it has not been feasible to use the two primer rolling circle amplification techniques with an acceptable level of specificity.
In work leading up to the present invention the inventors have determined the origin of and characterised this background amplification. This class of background amplification has been termed xe2x80x9cAmpXxe2x80x9d. The inventors have determined that it is an alternative amplification reaction which utilizes any linear nucleic acid probe molecules present in the reaction mixture. Typically the reaction products are multimers of head to tail tandem repeats. However, the inventors have determined that rather than encompassing sequence from the entire circular probe, the products of the AmpX reaction include repeats of a region of the linear target molecule that includes the two primer binding sites, the intervening sequence and some additional sequence of the template molecule flanking the primer binding sites.
Accordingly, the inventors have developed a method for minimizing AmpX background amplification by enriching for closed circular nucleic acid probe molecules prior to their amplification. By conducting the amplification step utilising an enriched population of closed circle nucleic acid probe molecules the incidence of background amplification caused by the AmpX reaction is significantly reduced, thereby enabling more specific rolling circle amplification to occur.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word xe2x80x9ccomprisexe2x80x9d, and variations such as xe2x80x9ccomprisesxe2x80x9d and xe2x80x9ccomprisingxe2x80x9d, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The subject specification contains nucleotide sequence information prepared using the programme PatentIn Version 2.0, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator  less than 210 greater than  followed by the sequence identifier (e.g.  less than 210 greater than 1,  less than 210 greater than 2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence are indicated by information provided in the numeric indicator fields  less than 211 greater than ,  less than 212 greater than and  less than 213 greater than , respectively. Nucleotide sequences referred to in the specification are defined by the information provided in numeric indicator field  less than 400 greater than  followed by the sequence identifier (e.g.  less than 4001 greater than 1,  less than 400 greater than 2, etc).
Accordingly, one aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
Another aspect of the present invention provides a method of rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
Still another aspect of the present invention more particularly provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
A further aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enzymatically enriching for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
Still a further aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
Yet another further aspect of the present invention provides a method for amplifying a circular nucleic acid probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising non-enzymatically enriching for said circular nucleic acid probe and subjecting said circular nucleic acid probe to amplification.
Still yet another further aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
Yet another aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
Yet a further aspect of the present invention provides a method of multiple primer rolling circle amplification comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a biotinylated capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
Another aspect, the present invention is directed to a method of enriching or a circular nucleic acid probe, said method comprising th e steps of facilitating g the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe.
Yet another aspect of the present invention provides a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment.
Still another aspect of the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; and generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment.
Still yet another aspect of the present invention is directed to a method of enriching for a circular nucleic acid probe, said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; and generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe.
In a further aspect there is provided in the method of amplifying a circular nucleic acid probe the improvement comprising amplifying a circular probe produced following interaction of a nucleic acid probe with a target nucleic acid sequence said method comprising enriching for said circular nucleic acid probe and then subjecting said circular nucleic acid probe to amplification.
In another further aspect there is provided in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
In yet another further aspect there is provided in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
In still yet another further aspect there is provided in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe by non-enzymatic enrichment; and subjecting said enriched circular nucleic acid probe to amplification.
In another aspect the present invention provides in the method of rolling circle amplification the improvement comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecule wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
Another aspect of the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid sequence; generating a circular nucleic acid probe and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
In another aspect the present invention contemplates a method of diagnosing a disease condition or detecting a genetic variant said method comprising the steps of facilitating the interaction of a nucleic acid probe with a target nucleic acid molecular wherein the terminal regions of said probe form non-contiguous duplexes; generating a circular nucleic acid probe, incorporating a capture ligand into the region intervening said terminal regions and enriching for said circular nucleic acid probe; and subjecting said enriched circular nucleic acid probe to amplification.
In still yet another aspect of the present invention is directed to a kit for facilitating rolling circle amplification said kit comprising compartments adapted to contain any one or more of nucleic acid probes, enzymes, capture ligands, means for isolating circular nucleic acid probes and reagents useful for facilitating circularisation, isolation and amplification of said probes. Further compartments may also be included, for example, to receive biological samples.